Evaluation of the biomarker candidate MFAP4 for non-invasive assessment of hepatic fibrosis in hepatitis C patients.

BACKGROUND: The human microfibrillar-associated protein 4 (MFAP4) is located to extracellular matrix fibers and plays a role in disease-related tissue remodeling. Previously, we identified MFAP4 as a serum biomarker candidate for hepatic fibrosis and cirrhosis in hepatitis C patients. The aim of the present study was to elucidate the potential of MFAP4 as biomarker for hepatic fibrosis with a focus on the differentiation of no to moderate (F0-F2) and severe fibrosis stages and cirrhosis (F3 and F4, Desmet-Scheuer scoring system). METHODS: MFAP4 levels were measured using an AlphaLISA immunoassay in a retrospective study including n = 542 hepatitis C patients. We applied a univariate logistic regression model based on MFAP4 serum levels and furthermore derived a multivariate model including also age and gender. Youden-optimal cutoffs for binary classification were determined for both models without restrictions and considering a lower limit of 80 % sensitivity (correct classification of F3 and F4), respectively. To assess the generalization error, leave-one-out cross validation (LOOCV) was performed. RESULTS: MFAP4 levels were shown to differ between no to moderate fibrosis stages F0-F2 and severe stages (F3 and F4) with high statistical significance (t test on log scale, p value <2.2·10(-16)). In the LOOCV, the univariate classification resulted in 85.8 % sensitivity and 54.9 % specificity while the multivariate model yielded 81.3 % sensitivity and 61.5 % specificity (restricted approaches). CONCLUSIONS: We confirmed the applicability of MFAP4 as a novel serum biomarker for assessment of hepatic fibrosis and identification of high-risk patients with severe fibrosis stages in hepatitis C. The combination of MFAP4 with existing tests might lead to a more accurate non-invasive diagnosis of hepatic fibrosis and allow a cost-effective disease management in the era of new direct acting antivirals.

MFAP4: a candidate biomarker for hepatic and pulmonary fibrosis?

BACKGROUND: Several comparable mechanisms have been identified for hepatic and pulmonary fibrosis. The human microfibrillar associated glycoprotein 4 (MFAP4), produced by activated myofibroblasts, is a ubiquitous protein playing a potential role in extracellular matrix (ECM) turnover and was recently identified as biomarker for hepatic fibrosis in hepatitis C patients. The current study aimed to evaluate serum levels of MFAP4 in patients with pulmonary fibrosis in order to test its potential as biomarker in clinical practice. A further aim was to determine whether MFAP4 deficiency in mice affects the formation of pulmonary fibrosis in the bleomycin model of lung fibrosis. METHODS: 91 patients with idiopathic pulmonary fibrosis (IPF), 23 with hypersensitivity pneumonitis (HP) and 31 healthy subjects were studied. In the mouse model, C57BL/6 Mfap4+/+ and Mfap4-/- mice between 6-8 weeks of age were studied. Serum levels of MFAP4 were measured by ELISA in patients and in mice. Surfactant protein D (SP-D) and LDH were measured as comparison biomarkers in patients with pulmonary fibrosis. Morphometric assessment and the Sircol kit were used to determine the amount of collagen in the lung tissue in the mouse model. RESULTS: Serum levels of MFAP4 were not elevated in lung fibrosis - neither in the patients with IPF or HP nor in the animal model. Furthermore no significant correlations with pulmonary function tests of IPF patients could be found for MFAP4. MFAP4 levels were increased in BAL of bleomycin treated mice with pulmonary fibrosis. CONCLUSIONS: MFAP4 is not elevated in sera of patients with pulmonary fibrosis or bleomycin treated mice with pulmonary fibrosis. This may be due to different pathogenic mechanisms of liver and lung fibrogenesis. MFAP4 seems to be useful as serum biomarker for hepatic but not for lung fibrosis.

Genome-wide analysis associates familial colorectal cancer with increases in copy number variations and a rare structural variation at 12p12.3.

Colorectal cancer (CRC) is one of the most common cancer worldwide. However, a large number of genetic risk factors involved in CRC have not been understood. Copy number variations (CNVs) might partly contribute to the 'missing heritability' of CRC. An increased overall burden of CNV has been identified in several complex diseases, whereas the association between the overall CNV burden and CRC risk is largely unknown. We performed a genome-wide investigation of CNVs on genomic DNA from 384 familial CRC cases and 1285 healthy controls by the Affymetrix 6.0 array. An increase of overall CNV burden was observed in familial CRC patients compared with healthy controls, especially for CNVs larger than 50kb (case/control ratio = 1.66, P = 0.025). In addition, we discovered for the first time a novel structural variation at 12p12.3 and determined the breakpoints by strategic PCR and sequencing. This 12p12.3 structural variation was found in four of 2862 CRC cases but not in 6243 healthy controls (P = 0.0098). RERGL gene (RERG/RAS-like), the only gene influenced by the 12p12.3 structural variation, sharing most of the conserved regions with its close family member RERG tumor suppressor gene (RAS-like, estrogen-regulated, growth inhibitor), might be a novel CRC-related gene. In conclusion, this is the first study to reveal the contribution of the overall burden of CNVs to familial CRC risk and identify a novel rare structural variation at 12p12.3 containing RERGL gene to be associated with CRC.

Hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome.

BACKGROUND: Hereditary nonpolyposis colorectal cancer HNPCC, Lynch syndrome) is a genetic disease of autosomal dominant inheritance. It is caused by a mutation in one of four genes of the DNA mismatch repair system and confers a markedly increased risk for various types of cancer, particularly of the colon and the endometrium. Its prevalence in the general population is about 1 in 500, and it causes about 2% to 3% of all colorectal cancers. Lynch syndrome is diagnosed in two steps: If it is suspected (because a patient develops cancer at an unusually young age or because of familial clustering), the tumor tissue is analyzed for evidence of deficient mismatch repair (microsatellite instability, loss of mismatch repair protein expression). If such evidence is found, a genetic mutation is sought. The identification of a pathogenic mutation confirms the diagnosis in the patient and enables predictive testing of other family members. Diagnostic evaluations for Lynch syndrome should be carried out with appropriate genetic counseling. METHOD: Selective literature review. RESULTS: Prospective cohort studies from Germany, Finland and the Netherlands have shown that colorectal cancers detected by systematic colonoscopic surveillance tend to be at an earlier stage than those that are discovered after the patients present with symptoms. The Finnish study also showed an overall reduction in cancer risk from colonoscopic polypectomy at regular intervals. CONCLUSION: The studies conducted so far have not yet clearly documented the putative benefit of an individualized, risk-adapted surveillance strategy. Until this is done, patients with Lynch syndrome and healthy carriers of causative mutations should be monitored with annual colonoscopy and (for women) annual gynecological examination.

Smad4 deficiency in cervical carcinoma cells.

Squamous cell carcinoma of the uterine cervix is one of the most frequent cancers affecting women worldwide. Carcinomas arise from cervical intraepithelial lesions, in which infection with high-risk human papillomavirus types has led to deregulated growth control through the actions of the viral E6 and E7 oncoproteins. The molecular mechanisms underlying progression to invasive tumor growth are poorly understood. One important feature, however, is the escape from growth inhibition by transforming growth factor beta (TGF-beta). Loss of chromosomal arm 18q is among the most frequent cytogenetic alterations in cervical cancers and has been associated with poor prognosis. Since the TGF-beta response is mediated by Smad proteins and the tumor suppressor gene Smad4 resides at 18q21, we have analysed the Smad4 gene for cervical cancer-associated alterations in cell lines and primary carcinomas. Here, we report Smad4 deficiency in four out of 13 cervical cancer cell lines which is due to an intronic rearrangement or deletions of 3' exons. All cell lines, however, showed either absent or moderate responsiveness to TGF-beta irrespective of their Smad4 status. In 41 primary squamous cervical carcinomas analysed, 10 samples showed loss of Smad4 protein expression and 26 samples a reduced expression. Altogether, our results strongly suggest that Smad4 gene alterations are involved in cervical carcinogenesis.

A polymorphism of the NFKBIA gene is associated with Crohn's disease patients lacking a predisposing allele of the CARD15 gene.

BACKGROUND AND AIMS: Nuclear factor kappa-B (NFkappaB) plays a crucial role in diseases associated with dysregulated immune response. NFkappaB inhibitor alpha downregulates the activity of NFkappaB. PATIENTS AND METHODS: To evaluate the contribution of the NFkappaB inhibitor alpha gene in Crohn's disease single nucleotide polymorphisms in the 3'-UTR and at position -420 in the promoter were studied in 259 patients with Crohn's disease genotyped for the variations of the CARD15 gene in comparison to 441 healthy controls. Additionally we screened the coding region of the NFkappaB inhibitor alpha gene for polymorphisms by SSCP analysis. RESULTS: In comparison to controls the A allele and the AA genotype frequencies of the single nucleotide polymorphisms in the 3'-UTR were significantly increased only in Crohn's disease patients without a variation in the CARD15 gene. Similarly, the difference between patients harboring no predisposing CARD15 alleles and patients harboring such a variation was significant. CONCLUSION: The findings indicate that the phenotype Crohn's disease is to be substructured with respect to genetic susceptibility.

Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experiments in healthy volunteers.

Glucagon-like peptide 1 (GLP-1) and analogues are being evaluated as a new therapeutic principle for the treatment of type 2 diabetes. GLP-1 suppresses glucagon secretion, which could lead to disturbances of hypoglycemia counterregulation. This has, however, not been tested. Nine healthy volunteers with normal oral glucose tolerance received infusions of regular insulin (1 mU x kg(-1) x min(-1)) over 360 min on two occasions in the fasting state. Capillary glucose concentrations were clamped at plateaus of 4.3, 3.7, 3.0, and 2.3 mmol/liter for 90 min each (stepwise hypoglycemic clamp); on one occasion, GLP-1 (1.2 pmol x kg(-1) x min(-1)) was administered i.v. (steady-state concentration, approximately 125 pmol/liter); on the other occasion, NaCl was administered as placebo. Glucagon, cortisol, GH (immunoassays), and catecholamines (radioenzymatic assay) were determined, autonomous and neuroglucopenic symptoms were assessed, and cognitive function was tested at each plateau. Insulin secretion rates were estimated by deconvolution (two-compartment model of C-peptide kinetics). At insulin concentrations of approximately 45 mU/liter, glucose infusion rates were similar with and without GLP-1 (P = 0.26). Only during the euglycemic plateau (4.3 mmol/liter), GLP-1 suppressed glucagon concentrations (4.1 +/- 0.4 vs. 6.5 +/- 0.7 pmol/liter; P = 0.012); at all hypoglycemic plateaus, glucagon increased similarly with GLP-1 or placebo, to maximum values greater than 20 pmol/liter (P = 0.97). The other counterregulatory hormones and autonomic or neuroglucopenic symptom scores increased, and cognitive functions decreased with decreasing glucose concentrations, but there were no significant differences comparing experiments with GLP-1 or placebo, except for a significant reduction of GH responses during hypoglycemia with GLP-1 (P = 0.04). GLP-1 stimulated insulin secretion only at plasma glucose concentrations of at least 4.3 mmol/liter. In conclusion, the suppression of glucagon by GLP-1 does occur at euglycemia, but not at hypoglycemic plasma glucose concentrations (< or = 3.7 mmol/liter). GLP-1 does not impair overall hypoglycemia counterregulation except for a reduction in GH responses, which is in line with other findings demonstrating pituitary actions of GLP-1. Below plasma glucose concentrations of 4.3 mmol/liter, the insulinotropic action of GLP-1 is negligible.